Antigen

Antigen Class

TFs and others

Antigen

Gata2

Cell type

Cell type Class

Blood

Cell type

Mast Cells

MeSH Description

Granulated cells that are found in almost all tissues, most abundantly in the skin and the gastrointestinal tract. Like the BASOPHILS, mast cells contain large amounts of HISTAMINE and HEPARIN. Unlike basophils, mast cells normally remain in the tissues and do not circulate in the blood. Mast cells, derived from the bone marrow stem cells, are regulated by the STEM CELL FACTOR.

Sample

source_name

BMMCs

strain

C57BL/6 * 129

cell type

Bone-marrow derived mast cells

chip antibody

Anti-GATA2, (Abcam, ab109241)

genotype

wild type

treatment

IgE anti_TNP and TNP-BSA

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Briefly, 1x10e7 BMMCs that were not treated or treated with IgE receptor crosslinking were fixed in complete medium with 1% Formaldehyde (Thermo Scientific, 28908). The fixation was quenched by adding glycine and then washed with ice-cold PBS. The cells were resuspended in lysis buffer with protease inhibitor cocktail (Sigma, SRE0055-1BO) and sonicated using Covaris S220 Focused-ultrasonicator (Covaris, Woburn, MA). The lysate was pre-cleared by incubating with protein A agarose/salmon sperm DNA slurry (Millipore, Cat# 16-157) and the supernatant was incubated with the anti-GATA2 antibody (ab109241, Abcam, Cambridge, MA) at 4 °C overnight. Then, the samples were incubated with salmon sperm DNA/protein A beads at 4 °C for one hour. The beads were washed using low salt immune complex wash buffer, high salt immune complex wash buffer once, LiCl immune complex wash buffer once and TE buffer twice. The beads were eluted with elution buffer. The crosslinking of eluted immunocomplexes was reversed by incubating with NaCl and RNase A (Roche, Cat# 11119915001) at 65 °C overnight, followed by incubation with Tris pH 6.5, EDTA pH 8.0, and Proteinase K at 55 °C for one hour. Finally, the recovered DNA was cleaned up using a QIAGEN QIAquick PCR purification kit (Qiagen, Valencia, CA). ChIP-seq library was prepared using TruSeq ChIP Library Preparation Kit (IP-202-1024, Illumina, San Diego, CA) according to the manufacturer's instructions. Briefly, 10 ng of ChIPed DNA was converted into blunt-ended fragments. A single adenosine nucleotide was added to the 3' ends of the blunt-ended fragments before indexing adaptors were ligated to the adenylated 3' ends. The ligated product was purified, size-selected and PCR amplified according to the manufacturer's instructions. The quality and quantity of the DNA library were assessed on an Agilent Technologies 2100 Bioanalyzer. The paired-ended sequencing was performed on an Illumina NovaSEQ6000 platform.

Sequencing Platform

instrument_model

Illumina NovaSeq 6000

mm10

Number of total reads

185766310

Reads aligned (%)

96.3

Duplicates removed (%)

22.8

Number of peaks

2015 (qval < 1E-05)

mm9

Number of total reads

185766310

Reads aligned (%)

96.2

Duplicates removed (%)

22.9

Number of peaks

1999 (qval < 1E-05)